Methods for producing carboxylate ligand modified ferric iron hydroxide colloids and related compositions and uses

ABSTRACT

Processes for recovering colloids of carboxylate ligand modified ferric iron hydroxide materials such as IHAT (Iron Hydroxide Adipate Tartrate) are described based on the use of water miscible non-aqueous solvents, such as ethanol, methanol and acetone. The processes produce materials with advantageous properties such as improved bioavailability, reduced aggregation and/or agglomeration and/or increased iron content.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry pursuant to 35 U.S.C. § 371of International Application No. PCT/EP2016/074022, filed Oct. 7, 2016,which claims the benefit of priority of GB Application No. 1517893.2,filed Oct. 9, 2015, each of which is incorporated by reference herein inits entirety for any purpose.

FIELD OF THE INVENTION

The present invention relates to methods for producing carboxylateligand modified ferric iron hydroxides, and in particular to methods forrecovering carboxylate ligand modified ferric iron hydroxide colloidsthat employ non-aqueous solvents. The present invention further relatesto iron supplements and compositions comprising carboxylate ligandmodified ferric iron hydroxides and their use in a method of treatingiron deficiency anaemia.

BACKGROUND OF THE INVENTION

Despite considerable global efforts with oral iron supplementation andfortification, iron deficiency remains the most common and widespreadnutritional disorder in the world. A key reason for this failure isthat, to address iron deficiency, oral iron supplementation needs to bewell tolerated, cheap, safe and effective. However, currently availablepreparations fail in at least one of these criteria. Simple ferrous iron[Fe(II)] salts are most commonly used as these are inexpensive and theiron is well absorbed. However, these are poorly tolerated and indeedappear to enhance systemic infection rates, may induce undesirablechanges to commensal bacteria of the colon and increase pro-inflammatorysignalling of the gut epithelium. Some forms of ferric iron [Fe(III)](e.g. ferric pyrophosphate) are considered safer and better tolerated inthe gut lumen than Fe(II), but have the disadvantage that they arepoorly absorbed.

As examples of prior art iron supplements, WO 2005/094203 (Navinta) andWO 2005/000210 (Chromaceutical) relate to processes for making sodiumferric gluconate complexes (Ferrlecit™) for use as an intravenouslyadministered iron supplements. These high molecular weight ironsaccharidic complexes are formed when the surface of freshlyprecipitated iron hydroxide particles are coated with gluconatemolecules, and subsequently form agglomerated mixtures of secondarycomplexes. US 2005/0256328 (Justus & Hanseler) also describe similarferric gluconate complexes for intravenous delivery. WO 2004/07444 andUS 2008/0274210 (Globoasia LLC) describe phosphate binding materialsbased on stoichiometric ferric citrate coordination complexes.

WO 2008/096130 (Medical Research Council) describes ferric ironoxo-hydroxide colloids that are modified synthetically so that dietarycarboxylic acid ligands are non-stoichiometrically incorporated into theiron oxo-hydroxide structure. These colloidal ligand modified ironoxo-hydroxides, in which the mineral phase is disrupted, mimic theferritin core—a natural dietary source of iron—and thus are wellabsorbed in humans with few or no side effects, providing a safe andefficacious oral iron supplement. The ligand modified ferricoxo-hydroxides described in WO 2008/096130 include nanoparticles of ironhydroxide modified with adipate (A) and tartrate (T) carboxylate ligandsin a 1:1:2 T:A:Fe molar ratio (Iron Hydroxide Adipate Tartrate or“IHAT”, seehttp://www.rsc.org/chemistryworld/2014/12/solving-iron-solubility-problem-profile-mrc).These materials are shown to be alternative safe iron delivery agentsand their absorption in humans correlated with serum iron increase(P<0.0001) and direct in vitro cellular uptake (P=0.001), but not withgastric solubility. IHAT also showed ˜80% relative bioavailability toFe(II) sulfate in humans and, in a rodent model, IHAT was equivalent toFe(II) sulfate at repleting haemoglobin. Furthermore, IHAT did notaccumulate in the intestinal mucosa and, unlike Fe(II) sulfate, promoteda beneficial microbiota. In cellular models, IHAT was 14-fold less toxicthan Fe(II) sulfate/ascorbate, itself has minimal acute intestinaltoxicity in cellular and murine models and shows efficacy at treatingiron deficiency anaemia (Pereira et al., Nanoparticulate iron(III)oxo-hydroxide delivers safe iron that is well absorbed and utilised inhumans, Nanomedicine, 10(8): 1877-1886, 2014). Other papers describingIHAT and its uses for treating iron deficiency include Aslam, et al.,Ferroportin mediates the intestinal absorption of iron from ananoparticulate ferritin core mimetic in mice (FASEB J. 28(8):3671-8,2014) and Powell et al., A nano-disperse ferritin-core mimetic thatefficiently corrects anaemia without luminal iron redox activity(Nanomedicine. 10(7):1529-38, 2014).

IHAT materials are produced in WO 2008/096130 by co-precipitating ferriciron ions and the organic acids by raising the pH of an aqueous solutionof the components from a pH at which they are soluble to a higher pH atwhich polymeric ligand modified ferric oxo-hydroxide forms. Theprecipitate is then dried, either by oven drying at 45° C. for 4-14 daysor freeze-drying at −20° C. and 0.4 mbar for a longer period, therebyproducing ligand modified ferric oxo-hydroxide suitable for formulationas an iron supplement. However, the success of IHAT as a widely usedsupplement means that there is a need in the art to improve the methodsused for the production of these materials, such that the materials areproduced cheaply at scale.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to improvements to methods forproducing carboxylate ligand modified ferric iron hydroxides, and in aparticular to methods for recovering and purifying carboxylate ligandmodified ferric iron hydroxide colloids. Generally, the carboxylateligands comprise one or more dicarboxylate ligands, such as tartrate,adipate and/or succinate.

Despite promising in vivo bioavailability and tolerability evidence, thelack of scalable and cheap manufacturing processes is an obstacle towidespread use of carboxylate ligand modified ferric iron oxo-hydroxidecolloids. Centrifugation or filtration could be used for recovery, butdue to the colloids' small size (Dv0.9 (i.e. 90%)<10 nm),ultracentrifugation or ultrafiltration would have to be employed, whichhave the disadvantage of being uneconomical at manufacturing scales.Therefore, the dry powders produced so far have been recovered by firstsynthesising a dispersion of small colloids and then evaporating thewater. However, this strategy requires a lengthy drying step (typicallyrequiring about a week at 45° C.), with the result that it is energyintensive and promotes unwanted particle agglomeration, that is part ofthe material may not re-disperse once back in water, reducing intestinalbioavailability. In addition, the prior art process leads to therecovery of soluble reaction products (e.g. NaCl) and unboundcarboxylate ligands with the iron colloids, and consequently the ironcontent in the powder (% w/w) is reduced as it is diluted by all theunused reactant materials. On the one hand, such low iron content isdisadvantageous in oral iron supplementation since large pill massesneed to be administered, which may impact negatively on patientcompliance. On the other hand, it might be expected that the unusedreactant materials contribute to the iron colloid particles remainingdisperse and thus facilitating the iron availability and absorption. Theideal situation would be to meet both of these conflicting goals byremoving unused reactant materials and retaining the particles ofcarboxylate ligand modified ferric iron hydroxides in a form in whichthey are bioavailable.

Surprisingly, we have found that ethanolic recovery of carboxylateligand modified ferric iron hydroxide colloid overcomes all of theseproblems and provides a rapid, cheap process to produce a dry powderfrom the synthesised iron material suitable for formulation. This drymaterial retains its colloidal properties and is sufficientlyconcentrated to be capable of being given as a single capsule, tablet orpowder for therapeutic supplementation, while removing unused reactantmaterials. The experiments described below also show that the materialsrecovered by using water miscible non-aqueous solvents such as ethanolhad appropriate dissolution rates, and in some cases dissolved morerapidly that the corresponding oven-dried materials. Other watermiscible non-aqueous solvents were found to be capable of providingsimilar results, in particular non-aqueous solvents such as methanol andacetone. The present inventors found that these methods producecarboxylate ligand modified ferric iron oxo-hydroxides materials havingsmaller primary particle sizes as compared to the prior art methodswhich are therefore more easily dissolved under lysosomal conditionswithin intestinal cells, and hence which have improved bioavailabilityvia oral delivery. It should be noted that these properties differ fromthe iron supplements for intravenous delivery, such as those disclosedin WO 2005/094203, WO 2005/000210 and US 2005/0256328, as theintravenous materials need to be sufficiently stable not to dissolverapidly in circulation as this would cause the significant patienttoxicity.

Additionally, as the methods lead to a reduction in the carboxylatecontent of the materials, the effect of this is to increase the overalliron content present in the materials on a percentage weight for weightbasis. Finally, despite the reduction in the carboxylate content, themethods help to produce materials in which aggregation and/oragglomeration of a fraction of the dried material is reduced.

Accordingly, in a first aspect, the present invention provides a methodof producing a carboxylate ligand modified ferric iron hydroxideformulation, the method comprising

-   -   mixing a colloidal suspension of the carboxylate ligand modified        ferric iron hydroxide in a water miscible non-aqueous solvent to        cause the carboxylate ligand modified ferric iron hydroxide to        agglomerate;    -   recovering the agglomerated carboxylate ligand modified ferric        iron hydroxide; and    -   drying the carboxylate ligand modified ferric iron hydroxide to        produce the carboxylate ligand modified ferric iron hydroxide        formulation, wherein the carboxylate ligand comprises one or        more dicarboxylate ligands.

Preferably, the water miscible non-aqueous solvent is selected fromethanol, methanol and/or acetone.

The method may optionally comprise the further step of carboxylateligand modified ferric iron hydroxide formulation in a tablet or acapsule for oral delivery.

In a further aspect, the present invention provides an iron supplementtablet, capsule or powder comprising a carboxylate ligand modifiedferric iron hydroxide composition as obtainable by the method describedherein.

In a further aspect, the present invention provides a carboxylate ligandmodified ferric iron hydroxide material having a three dimensionalpolymeric structure in which the carboxylate ligands arenon-stoichiometrically substituted for the oxo or hydroxy groups of theferric iron hydroxide so that some of the ligand integrates into thesolid phase by formal metal-ligand bonding, wherein the threedimensional polymeric structure of the carboxylate ligand modifiedferric iron hydroxide is such that the substitution of the oxo orhydroxy groups by the carboxylate ligands is substantially random,and/or wherein on dispersion in water the material produces amicroparticulate ferric iron fraction comprising less than 3.0% of thetotal ferric iron present in the material.

In a further aspect, the present invention provides an iron supplementtablet, capsule or powder comprising a carboxylate ligand modifiedferric iron hydroxide composition or a carboxylate ligand modifiedferric iron hydroxide material of the present invention for use in amethod of treating iron deficiency anaemia, iron deficiency and anaemiaof chronic disease. These materials and compositions are preferablyformulated for oral delivery.

Embodiments of the present invention will now be described by way ofexample and not limitation with reference to the accompanying figures.However various further aspects and embodiments of the present inventionwill be apparent to those skilled in the art in view of the presentdisclosure.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Size distribution of an IHAT suspension (produced as per Example1).

FIG. 2. X-ray diffractogram of an IHAT powder recovered by oven drying(as per Example 2). Top trace shows full scale with fitting of NaCl andKCl traces (key peaks indicated by vertical lines starting at the xaxis). Bottom trace shows zoomed XRD pattern of disrupted ferrihydritebaseline, showing the presence of sharp diffraction peaks frompotassium/sodium chloride and sodium adipate plus an underlying, broad,diffuse peak at ca. 33° 2-theta that is consistent with a modifiedferrihydrite.

FIG. 3. X-ray diffractogram of an IHAT powder in which unbound ligandsand reaction products (e.g. NaCl) were removed by ultrafiltration (asper Example 3). Trace shows the presence of one very broad and diffusediffraction ‘peak’ centred around 32 to 33° 2-theta) that is consistentwith a modified ferrihydrite.

FIG. 4. X-ray diffractogram of an IHAT powder produced after ethanolicrecovery (as per Example 4) showing reduction, but not elimination, ofNaCl and KCl traces relative to regular IHAT (shown in FIG. 2). Key NaCland KCl peaks are indicated by vertical lines starting at the x axis.

FIG. 5. Overlay of ferrihydrite XRD baselines for ultra-filtered (top),ethanolic recovered (middle) and regular (bottom) IHAT powders. Whilstall three spectra show a level of ferrihydrite disruption, ethanolicrecovery resulted in additional disruption and reduction of crystallitesize, as demonstrated by the attenuation of the ferrihydrite peak.

FIG. 6. Bright field analysis (top, right and left), diffraction pattern(bottom left) and spot EDX (bottom right) of ethanolic recovered IHAT(as per Example 4). Bright field analysis shows agglomerates of veryfine-grained nanocrystalline material giving two broad selected areaelectron diffraction rings centred around 0.29 and 0.15 nm consistentwith the main broad peak identified by XRD. The corresponding EDXspectrum shows Fe, O plus C, Na, K and Cl. Overall, these findings areconsistent with a ligand-modified ferrihydrite.

FIG. 7. Fe, O and C EELS edges (x-axis is in eV) of area shown in FIG. 6and TEM image (bottom right) of the beam altered specimen after EELanalysis. EEL spectra are consistent with prior ligand modifiedferrihydrite spectra for material collected by oven drying (ferric ironplus organic ligand-altered carbon and oxygen edges, see Pereira et al.,Nanomedicine, 10(8): 1877-1886, 2014) and the TEM image recorded afterthe prolonged exposure required to collect EEL spectra confirms thenanocrystalline nature of the specimen upon beam damage (again aspreviously seen for material collected by oven drying; Pereira et al.,Nanomedicine, 10(8): 1877-1886, 2014)

FIG. 8. In vitro lysosomal dissolution over time of regular (top),ethanolic recovered (middle) and ultra-filtered (bottom) IHAT materials.

FIG. 9. Size distribution of IHAT re-suspended in water to originalconcentration after oven drying (as per Example 2) showing presence ofagglomerates (top). Removal of aggregates by centrifugation enables sizedetermination of the colloidal fraction (bottom).

FIG. 10. Phase distribution after resuspending ultra-filtered, ethanolicrecovered or oven dried (i.e. regular) IHAT to their original ironconcentrations. The three dry materials were produced from the same IHATsuspension (40 mM Fe; pH 7.42).

FIG. 11. Size distribution of ethanolic recovered IHAT (example 4) thatwas re-suspended in water to its original concentration (40 mM). Notethat upon resuspension agglomerates were absent, unlike in FIG. 8. Eachindividual trace corresponds to an analytical replicate (N=3).

FIG. 12. Size distribution (mean DV_(0.5)=4.0 nm) of acetonic recoveredIHAT (Example 6) re-suspended in water back to 40 mM Fe. Each individualtrace corresponds to an analytical replicate (N=3).

FIG. 13. Size distribution (mean DV_(0.5)=4.8 nm) of methanolicrecovered IHAT (Example 5) re-suspended in water back to 40 mM Fe. Eachindividual trace corresponds to an analytical replicate (N=3).

FIG. 14. TEM images of IHAT recovered using (a) ethanolic treatment, (b)oven drying and (c) ultrafiltration. The images show that the ethanolicrecovered material was the finest grained and hence the most amorphousand with a smaller crystallite size. The materials recovered by ovendrying and ultrafiltration were indistinguishable.

FIG. 15. Phase distribution of concentrated ethanolic IHAT before(“original”; as per Example 7) and after ethanolic recovery(“resuspended”; as per Example 8). Experimental details are provided inExample 9.

FIG. 16. Size distribution of concentrated ethanolic IHAT before(“original”; as per Example 7) and after ethanolic recovery(“resuspended”; as per Example 8). Experimental details are provided inExample 9. Each individual trace corresponds to the average of threeanalytical replicates (N=3; standard deviation bars shown).

DETAILED DESCRIPTION

Production of Carboxylate Ligand Modified Ferric Iron Hydroxides

The carboxylate ligand modified ferric iron hydroxides may be producedunder specific conditions by dissolving a suitable ferric iron [Fe(III)]salt and then inducing the formation of polymeric iron hydroxides inwhich a proportion of the carboxylate ligands become integrated into thesolid phase through formal metal-iron (M-L) bonding, i.e. not all of theligand (L) is simply trapped or adsorbed in the bulk material. Thebonding of the metal ion in the materials can be determined usingphysical analytical techniques such as X-ray diffraction (XRD), whichdemonstrates disruption of mineral phase, i.e. with peak shifts and bandbroadening due increased amorphousness resulting from ligandincorporation in the primary particle.

In the carboxylate ligand modified iron hydroxides disclosed herein, thepresence of formal metal ion-ligand bonding is one feature thatdistinguishes the materials from other products such as “ironpolymaltose” (Maltofer) in which particulate crystalline iron hydroxideis surrounded by a sugar shell formed from maltose and thus is simply amixture of iron oxo-hydroxide and sugar at the nano-level (Heinrich(1975); Geisser and Müller (1987); Nielsen et al (1994; U.S. Pat. No.3,076,798); US2006/0205691).

In addition, the carboxylate ligand modified ferric iron hydroxides ofthe present invention are solid phase metal poly oxo-hydroxides modifiedby non-stoichiometric ligand incorporation. This distinguishes them fromthe numerous metal-ligand classical coordination complexes that are wellreported in the art (WO 03/092674, WO 06/037449) which arestoichiometric. Although generally soluble, such complexes can beprecipitated from solution at the point of supersaturation, for exampleferric trimaltol, Harvey et al. (1998), WO 03/097627; ferric citrate, WO04/074444, US 2008/0274210 and ferric tartrate, Bobtelsky and Jordan(1947) and, on occasions, may even involve stoichiometric binding ofhydroxyl groups (for example, ferric hydroxide saccharide, U.S. Pat. No.3,821,192).

Without modification, the primary particles of the carboxylate ligandmodified ferric iron hydroxides used herein have ferric iron oxide coresand ferric hydroxide surfaces and within different disciplines may bereferred to as metal oxides or metal hydroxides. The use of the term‘oxo-hydroxy’ or ‘oxo-hydroxide’ may be used interchangeably and isintended to recognise these facts without any reference to proportionsof oxo or hydroxy groups. As described herein, the carboxylate ligandmodified ferric iron hydroxides of the present invention are altered atthe level of the primary particle of the metal hydroxide with at leastsome of the ligand being introduced into the structure of the primaryparticle, i.e. leading to doping or contamination of the primaryparticle by the ligand. This may be contrasted with the formation ofnano-mixtures of metal oxo-hydroxides and an organic molecule, such asiron saccharidic complexes, in which the structure of the core is not soaltered.

The primary particles of the carboxylate ligand modified ferric ironhydroxides materials described herein are generally produced byprecipitation. The use of the term “precipitation” often refers to theformation of aggregates or agglomerates of materials that do separatefrom solution by sedimentation or centrifugation. Here, the term“precipitation” is intended to describe the formation of all solid phasematerial, including agglomerates or other solid phase materials thatremain as non-soluble moieties in suspension, whether or not they beparticulate, colloidal or sub-colloidal and/or nanoparticulates or yetsmaller clusters.

In the present invention, reference may be made to the carboxylateligand modified ferric iron hydroxides having three dimensionalpolymeric structures that generally form above the criticalprecipitation pH. As used herein, this should not be taken as indicatingthat the structures of the materials are polymeric in the strict senseof having a regular repeating monomer unit because, as has been stated,ligand incorporation is, except by co-incidence, non-stoichiometric.Without wishing to be bound by any particular theory, the inventorsbelieve that the carboxylate ligand species is introduced into the solidphase structure by substituting for oxo or hydroxy groups of the formingtwo dimensional iron oxo-hydroxide chains which then cross-link to formthree dimensional structures and so the ligand leads to a change insolid phase order. In some cases, for example the production of theferric iron materials exemplified herein, the ligand species may beintroduced into the solid phase structure by the substitution of oxo orhydroxy groups by ligand molecules in a manner that decreases overallorder in the solid phase material. While this still produces solidcarboxylate ligand modified ferric iron hydroxides that in the grossform have one or more reproducible physicochemical properties, thematerials have a more amorphous nature compared, for example, to thestructure of the corresponding unmodified metal oxo-hydroxide. Thepresence of a more disordered or amorphous structure can readily bedetermined by the skilled person using techniques well known in the art.One exemplary technique is transmission electron microscopy (TEM). Highresolution transmission electron microscopy allows the crystallinepattern of the material to be visually assessed. It can indicate theprimary particle size and structure (such as d-spacing), give someinformation on the distribution between amorphous and crystallinematerial, and show that the material possesses a structure consistentwith a 2-line ferrihydrite-like structure even when modified. Using thistechnique, it is apparent that the chemistry described above increasesthe amorphous phase of materials described herein compared tocorresponding materials without the incorporated ligand. This may beespecially apparent using high angle annular dark fieldaberration-corrected scanning transmission electron microscopy due tothe high contrast achieved while maintaining the resolution, thusallowing the surface as well as the bulk of the primary particles of thematerial to be visualised.

Additionally or alternatively, upon ligand modification, the kinetics ofdissolution of the carboxylate ligand modified ferric iron hydroxidesare accelerated, for example as illustrated in the lysosomal assay,compared to the corresponding materials without the incorporated ligand.Examples of the properties that can be usefully modulated for materialsused for iron supplementation or fortification include: dissolution(rate and pH dependence), adsorption and absorption characteristics,reactivity-inertness, melting point, temperature resistance, particlesize, surface charge, density, light absorbing/reflecting properties,compressibility, colour and encapsulation properties. Examples ofproperties that are particularly relevant to the field of supplements orfortificants are physicochemical properties selected from one or more ofa dissolution profile, an adsorption profile or a reproducible elementalratio. In this context, a property or characteristic may be reproducibleif replicate experiments for ethanolic recovery are reproducible withina standard deviation of preferably ±20%, and more preferably ±10%, andeven more preferably within a limit of ±5%.

The dissolution profile of the solid ligand-modified poly oxo-hydroxymetal ion materials can be represented by different stages of theprocess, namely dispersion or re-suspension. The term dissolution isused to describe the passage of a substance from solid to soluble phase.

In the present invention, the carboxylate ligand modified ferric ironhydroxide materials described herein differ from prior art materials,for example IHAT produced by oven-drying or ultrafiltration, in havingimproved dispersion properties when the materials are resuspended inwater. This can be assessed using the protocol described in the examplesbelow in which a homogeneous aliquot of a suspension of the materials istaken and then the soluble, nanoparticulate and microparticulate ferriciron fractions separated by centrifugation (microparticulate fractionsediments) and ultrafiltration (soluble phase passes the filter). Anycentrifugable phase formed may be separated from the solution bycentrifugation (e.g. for 10 minutes at 13000 rpm; benchtop centrifuge).The iron concentration in the supernatant fraction may be determined byinductively coupled plasma optical emission spectrometry (ICP-OES). Todifferentiate between soluble iron and colloidal iron (non-centrifugableparticles) in the supernatant, ultrafiltration may be used for exampleusing a Vivaspin 3,000 Da molecular weight cut-off polyethersulfonemembrane and the fraction again analysed by ICP-OES.

After they have been dried, when the carboxylate ligand modified ferriciron hydroxides materials of the present invention are dispersed inwater at 40 mM Fe they produce small amounts or substantially nomicroparticulate fraction, with the ferric iron phase distribution beingbetween soluble material and a nanoparticulate fraction, for examplethey will contain a microparticulate ferric iron fraction that has lessthan 5.0%, 4.0% 3.0%, 2.0%, 1.5%, 1.0%, 0.5% or 0.25% of the total ironpresent in the materials, and preferably substantially nomicroparticulate iron. This may be contrasted with the correspondingoven dried materials which disperse to produce a microparticulate ferriciron fraction containing about 5 to 10% of the total iron content.

Accordingly, the present invention provides a carboxylate ligandmodified ferric iron hydroxide material having a three dimensionalpolymeric structure in which the carboxylate ligands arenon-stoichiometrically substituted for the oxo or hydroxy groups of theferric iron hydroxide so that some of the ligand integrates into thesolid phase by formal metal-ligand bonding, wherein the threedimensional polymeric structure of the carboxylate ligand modifiedferric iron hydroxide is such that the substitution of the oxo orhydroxy groups by the carboxylate ligands is substantially random, andwherein on dispersion in water the material produces a microparticulateferric iron fraction comprising less than 3% of the total ferric ironpresent in the material.

In the carboxylate ligand modified iron hydroxides produced by themethods disclosed herein, the carboxylate ligands may be one, two, threeor four or more carboxylate ligands in the form of the carboxylate ionor the corresponding carboxylic acid. Generally, the ligand is adicarboxylic acid ligand, and may be represented by the formulaHOOC—R₁—COOH (or an ionised form thereof), where R₁ is an optionallysubstituted C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl or C₁₋₁₀ alkynyl group. The useof ligands in which R₁ is a C₁₋₁₀ alkyl group, and more preferably is aC₂₋₆ alkyl group, is preferred. Preferred optional substituents of theR₁ group include one or more hydroxyl groups, for example as present inmalic acid. These ligands include carboxylic acids such asadipate/adipic acid, tartrate/tartaric acid, glutarate/glutaric acid,malate/malic acid, succinate/succinic acid, aspartate/aspartic acid,pimelate/pimelic acid, citrate/citric acid, lactate/lactic acid orbenzoate/benzoic acid. In the production of some preferred materials,such as IHAT, two different ligands are used, such as adipate/adipicacid and tartrate/tartaric acid. Other examples of preferredcombinations of ligands include tartrate/tartaric acid andsuccinate/succinic acid. Particularly preferred materials are formedusing the following molar ratios of ligands and Fe(III):

Molar Ratio Material Ligands ligand:Fe Nano Fe(III) (a) Tartaric acid(T) 1:1:2 (T:A:Fe) “IHAT” Adipic acid (A) Nano Fe(III) (b) Tartaric acid(T) 1:1:2 (T:S:Fe) Succinic acid (S) Nano Fe(III) (c) Tartaric acid (T)1:6:2 (T:S:Fe) Succinic acid (S)

Without wishing to be bound by any particular theory, the presentinventors believe that in IHAT it is the tartrate/tartaric acid ligandsthat are mostly responsible for the disruption of the iron hydroxidestructure of the primary particles (Nanomedicine, 10(8): 1877-1886,2014). In view of this observation, in a further embodiment, thecarboxylate ligand modified iron hydroxides may be modified bytartrate/tartaric acid as the sole carboxylate ligand.

The ratio of the ferric iron ion(s) to the carboxylate ligands can bevaried according to the methods disclosed herein and may vary one ormore properties of the materials. Generally, the useful ratios of M:Lwill be between 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1 and 1:2, 1:3, 1:4, 1:5or 1:10, and preferably between 4:1 and 1:1. By way of example, in thepreferred IHAT materials, the concentration of ferric iron ions may bebetween 20 mM and 80 mM, the concentration of adipate is between 10 mMand 40 mM and the concentration of tartrate is between 10 mM and 40 mM.In the synthesis of IHAT, a concentration of ferric iron of about 40 mMwas used with 20 mM adipic acid and 20 mM tartaric acid. Alternatively,and in particular where different ratios of the components are used, theconcentration of ferric iron may be between 20 mM and 500 or 1000 mM,the concentration of adipate may be between 10 mM and 150 mM and theconcentration of tartrate may be between 10 mM and 250 mM.

In the case of materials using tartrate/tartaric acid as the solecarboxylate ligand, or where adipate is capped at its maximum aqueousconcentration (e.g. 150 mM at room temperature), a higher concentrationof ferric iron ions may be used between a lower limit 80 mM, 100 mM and120 mM and an upper limit of 250 mM, 350 mM, 500 mM and 1000 mM,optionally in combination with a concentration of tartrate/tartaric acidbetween 20 mM and 250 mM or 500 mM.

The present invention may employ any way of forming hydroxide ions atconcentrations that can provide for hydroxy surface groups and oxobridging in the formation of the carboxylate ligand modified ferric ironhydroxide materials. Examples include but are not limited to, alkalisolutions such as sodium hydroxide, potassium hydroxide and sodiumbicarbonate.

The methods of the present invention produce a suspension of particleshaving a size distribution (as a percentage of particle volume) betweenabout 1 nm and 20 nm in diameter, with the majority of the particles (ina volume-based distribution) having a size distribution between about 2nm and 10 nm in diameter. Within a given size range, it is preferredthat at least 75% of the nanoparticles of carboxylate ligand modifiedferric iron hydroxide have an average diameter in the range, and morepreferably that at least 90% of the nanoparticles of carboxylate ligandmodified ferric iron hydroxide have an average diameter in the range.The hydrodynamic particle size of colloidal suspensions may bedetermined by dynamic light scattering (DLS), for example using aZetasizer Nano-ZS (Malvern Instruments, UK). The reduction incrystallite size in non-aqueous solvent recovered materials is toosubtle to be determined by dynamic light scattering since this techniquealso captures size of the surrounding water shell in the redispersedparticles. Instead, TEM or XRD (attenuation and/or shift of theferrihydrite band) should be employed.

The exact conditions of mixing and precipitation of the carboxylateligand modified ferric iron hydroxides will vary depending upon thedesirable characteristics of the solid material. Typical variables are:

(1) Starting pH (i.e. the pH at which metal ion and ligand species aremixed). This will generally be a different pH to that at which hydroxypolymerisation commences. Preferably, it is a more acidic pH, morepreferably below a pH of 2.

(2) The pH at which polymerisation of the carboxylate ligand modifiedferric iron hydroxide commences. This is always a different pH to thatof the starting pH. Preferably, it is a less acidic pH and mostpreferably above a pH of 1.5 or 2.

(3) Final pH. This will always promote precipitation and may promoteagglomeration of the carboxylate ligand modified ferric iron hydroxidesand preferably will be a higher pH than the pH at which hydroxypolymerisation commences. In this case, a final pH between pH 7.0 and9.0, and more specifically between pH 7.4 and 8.5 is preferred.(4) Rate of pH change from commencement of polymerisation of thecarboxylate ligand modified ferric iron hydroxide to completion ofreaction. This will occur within a 24 hour period, preferably within anhour period and most preferably within 20 minutes.(5) Concentrations of metal ions and ligand species. While theconcentration of OH is established by the pH during hydroxypolymerisation, the concentrations of total metal ion and total ligandspecies in the system will be fixed by the starting amounts in themixture and the final solution volume. Typically, this will exceed 10⁻⁶molar for both metal ion and ligand species and more preferably it willexceed 10⁻³ molar. Concentrations of metal ion and ligand species areindependent and chosen for one or more desired characteristics of thefinal material.(6) Solution phase. The preferred solution for this work is aqueous andmost preferably is water.(7) Temperature. The preferred temperature is above 0 and below 100° C.,typically between room temperature (20-30° C.) and 50° C. or 100° C.,most typically at room temperature.(8) Ionic strength. Electrolyte such as, but not limited to, potassiumchloride and sodium chloride, may be used in the procedure. The ionicstrength of the solution may thus range from that solely derived fromthe components and conditions outlined in (1)-(8) above or from thefurther addition of electrolyte which may be up to 10% (w/v), preferablyup to 2%, and most preferably <1%.

After separation of the precipitated material, it may optionally bedried before use of further formulation. The dried product may, however,retain some water and be in the form of a hydrated carboxylate ligandmodified ferric iron hydroxide. It will be apparent to those skilled inthe art that at any of the stages described herein for recovery of thesolid phase, excipients may be added that mix with the carboxylateligand modified ferric iron hydroxides but do not modify the primaryparticle and are used with a view to optimising formulation for theintended function of the material.

Purification and Recovery of the Carboxylate Ligand Modified Ferric IronHydroxides

The methods of the present invention enable the large scale productionof carboxylate ligand modified ferric iron hydroxide formulation, andespecially one in which the iron content is greater than that producedwhen oven drying is used. The methods also enable the carboxylate ligandmodified ferric iron hydroxide to be separated from unreacted startingmaterials, such as free unreacted ligand, unreacted ferric iron ions,sodium ions, potassium ions and/or chloride ions, and by-products suchas salts, which is not possible in the prior art oven drying methods.

The carboxylate ligand modified ferric iron hydroxide of the presentinvention generally have an iron content of at least 10% Fe (w/w), andmay have an iron content of at least 15% Fe (w/w), or an iron content ofat least 20% Fe (w/w), or an iron content of at least 25% Fe (w/w), oran iron content of at least 30% Fe (w/w). Lower levels of iron contentare generally due to the presence of excess unreacted ligand or saltresulting from the synthesis of the materials. The choice of the ironcontent of the final formulation that includes the carboxylate ligandmodified ferric iron hydroxide will be dependent on a range of factors,and a higher iron content as compared to oven dried materials may beadvantageous in helping to reduce the size of tablets or capsulescontaining the carboxylate ligand modified ferric iron hydroxide, e.g.for improving ease of administration or helping with patient compliance.It will be obvious to those in the art that excipients including thesame or different ligand(s) as in the synthesis could be mixed with thefinal product to provide advantageous formulation properties such as,but not restricted to, the prevention of aggregation or agglomeration orto alter powders flow properties in manufacturing or to aid tableting inmanufacture.

As stated above, the methods of the present invention involve mixing acolloidal suspension of the carboxylate ligand modified ferric ironhydroxide with a water miscible non-aqueous solvent, generally ethanol,methanol or acetone, or mixtures thereof. In a preferred embodiment, thewater miscible non-aqueous solvent is ethanol. Conveniently, the ratioof the volume of the water miscible non-aqueous solvent to the colloidalsuspension of the carboxylate ligand modified ferric iron hydroxide isbetween 1:1 and 5:1. The present inventors found that the addition ofwater miscible non-aqueous solvents causes the carboxylate ligandmodified ferric iron hydroxide to agglomerate enabling it to beseparated from excess reaction products such as non-incorporated ligandas described above and then recovered, for example by centrifugation orfiltration. After the material has been recovered, it may be dried. Inthe drying step, the time and/or temperature used are generally shorterand lower than the prior art oven drying and preferably the drying steptakes 24 hours or less at 45° C. Other examples of water misciblenon-aqueous solvents are described athttps://en.wikipedia.org/wiki/List_of_water-miscible_solvents.

Accordingly, in some aspects, the present invention may use a watermiscible non-aqueous solvent, or mixtures thereof, other than ethanol,methanol and/or acetone, and especially water miscible non-aqueoussolvent that are non-toxic or Generally Regarded As Safe (G.R.A.S.).This means that the water miscible non-aqueous solvents include:acetone, acetonitrile, butanol, 1,2-butanediol, 1,3-butanediol,1,4-butanediol, diethanolamine, diethylenetriamine, dimethyl sulfoxide,ethanol, ethylamine, ethylene glycol, glycerol, methanol, methyldiethanolamine, 1-propanol, 1,3-propanediol, 1,5-pentanediol,2-propanol, propylene glycol and triethylene glycol, and mixturesthereof.

A further advantage of the present invention is that the carboxylateligand modified ferric iron hydroxides are generally at least asamorphous and crystallite size at least as small as prior art materialsbecause rapid drying of the water miscible non-aqueous solvent shortensthe ageing process of a colloid that would otherwise occur if an aqueoussuspension is dried using the slower approaches of the prior art. Ingeneral, these ageing processes increase the crystallinity or increasecrystallite size or reduce the amorphousness of the materials. Withoutwishing to be bound by any particular theory, the present inventorsbelieve that amorphous carboxylate ligand modified ferric ironhydroxides are required for good oral bioavailability. FIG. 14 showsthat carboxylate ligand modified ferric iron hydroxides using watermiscible non-aqueous solvent was more finely grained, and hence moreamorphous, than the materials recovered using oven drying orultrafiltration. The materials of the present invention are thereforelikely to have improved in vivo bioavailability as compared to the priorart materials produced using oven drying or ultrafiltration.

After drying, the carboxylate ligand modified ferric iron hydroxideformulation will generally have a mean particle size between 1 and 20nm, and more preferably between 1 and 10 nm.

Prior to formulating the carboxylate ligand modified ferric ironhydroxide, e.g. in a form for oral delivery, the method may comprise oneor more additional processing steps. Examples of these include millingor micronizing the carboxylate ligand modified ferric iron hydroxidecomposition. The carboxylate ligand modified ferric iron hydroxidecomposition may then be mixed with one or more pharmaceuticallyacceptable excipients and then formed in a final form for oral delivery,for example by making tablets or capsules.

Formulations and Uses

The carboxylate ligand modified ferric iron hydroxides produced by themethods of the present invention may be formulated for use assupplements, and especially as therapeutic iron supplements. This meansthat the formulations may be mixed with one or more pharmaceuticallyacceptable excipients, carriers, buffers, stabilisers or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the carboxylateligand modified ferric iron hydroxides for iron supplementation.

The precise nature of the carrier or other component may be related tothe manner or route of administration of the composition, in the presentcase generally via gastrointestinal delivery, in particular oraldelivery. Pharmaceutical compositions for oral administration may be intablet, capsule, powder, gel or liquid form. In some instances, thematerials may be directly orally taken, while in other embodiments, theymay be provided in a form suitable for mixing with food or drink andtaken in this manner. The latter may be termed fortification but theterms supplement and supplementation are herein included to cover thisas well as usual supplement practice.

Tablets are formed by compressing an active substance with components toenable the formation of the tablet and its dissolution after it has beentaken by a subject. Accordingly, a tablet may include a solid carrier,such as gelatin or an adjuvant or carrier, a compressibility agentand/or a flowing agent. In the present invention, an iron supplement inthe form of a tablet may comprise one or more of the carboxylate ligandmodified ferric iron hydroxides (for example forming 5-60% (w/w) of thetablet) and one or more fillers, disintegrants, lubricants, glidants andbinders (for example forming the remaining 40-95% (w/w) of the tablet).In addition, the tablet may optionally comprise one or more coatings,for example to modify dissolution of the tablet for either quick orsustained release, and/or one of more coatings to disguise the taste ofthe tablet or to make it easier for a subject to take orally.

Generally, capsules are formed by enveloping an active substance in agelatinous envelope. As with tablets, capsules may be designed for quickor sustained release depending on the properties of the envelope or acoating provided on it. Release of the active substance may also becontrolled by modifying the particle size(s) of the active substancecontained with the envelope. Capsules are generally either hard shelledor soft shelled. Hard shelled capsules are typically made using gelatinto encapsulate the active substance and may be formed by processes suchas extrusion or spheronisation. Hard shelled capsules may be formed bysealing together two half shells to form the final capsule. Soft shelledcapsules are generally formed by suspending an active ingredient in oilor water and then forming the envelope around the drops of the liquid.Other components of capsules include gelling agents, plantpolysaccharides, plasticizers, e.g. for modulating the hardness of thecapsule, colouring agents, preservatives, disintegrants, lubricants andcoatings.

The carboxylate ligand modified ferric iron hydroxides used inaccordance with the present invention that are to be given to anindividual are preferably administered in a “prophylactically effectiveamount” or a “therapeutically effective amount” (as the case may be,although prophylaxis may be considered therapy), this being sufficientto show benefit to the individual (e.g. bioavailability). The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of what is being treated. Prescriptionof treatment, e.g. decisions on dosage etc., is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams &Wilkins. A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially, dependent uponthe condition to be treated.

By way of example, iron supplements are generally administered at dosesof between 100 mg Fe to 250 mg Fe per day, and often at doses between 50mg Fe and 80 mg Fe (e.g. about 60 mg Fe) three times a day (t.d.s.).Single dosing may be possible using a sustained release formulation.Prophylactic supplementation may use lower doses, but it is desirable tohave any dose containing as high a percentage of the active agent (iron)as possible as this will minimise the size of the dose (capsule, pilletc.). In this aspect, this invention minimises non-active ingredients,such as unreacted ligands, of the formulation and allows the active ironmaterial to be well concentrated in the oral delivery dose.

The carboxylate ligand modified ferric iron hydroxides may be used assupplements for nutritional or medical benefit. In this area, there arethree main examples:

(i) Therapeutic (prescription) supplements, which are generallyadministered orally for the treatment of indications including irondeficiency anaemia, iron deficiency and anaemia of chronic disease. Thetherapeutic administration of carboxylate ligand modified ferric ironhydroxides of the present invention may be in conjunction with othertherapies, for example with the concomitant use of erythropoietin.(ii) Nutritional supplements (self prescribed/purchased supplements)which are usually for oral delivery.(iii) Fortificants. These may be traditional forms—in terms of beingadded to food prior to purchase—or more recent fortificant forms such as‘Sprinkles’ which are added (like salt or pepper) to food at the time ofingestion.

In all formats, but most especially for fortificants, subsequentformulation, such as addition of a protective coating (e.g. lipid), maybe necessary to make the material compatible with its intended usage. Inaddition, any of these supplemental forms can be co-formulated, eitherby incorporation within the material through use of co-formulatedmaterial(s) as ligand(s) or through trapping/encapsulation of saidmaterials, or simply through co-delivery of said materials.

As described herein, one particular application of the carboxylateligand modified ferric iron hydroxides of the present invention is forthe treatment of mineral deficiencies, for example iron deficiency.

By way of example, the carboxylate ligand modified ferric ironhydroxides disclosed herein may be used to deliver iron to an individualfor use in the prophylaxis or treatment of iron deficiency or irondeficiency anaemia which may be suspected, or diagnosed through standardhaematological and clinical chemistry techniques. Iron deficiency andiron deficiency anaemia may occur in isolation, for example due toinadequate nutrition or due to excessive iron losses, or they may beassociated with stresses such as pregnancy or lactation, or they may beassociated with diseases such as inflammatory disorders, cancers andrenal insufficiency. In addition, there is evidence that the reducederythropoiesis associated with anaemia of chronic disease may beimproved or corrected by the effective delivery of systemic iron andthat co-delivery of iron with erythropoietin or its analogues may beespecially effective in overcoming reduced erthropoietic activity. Thus,by way of further example, the ferric iron compositions disclosed hereinmay be used to deliver iron to an individual for use in the treatment ofsub-optimal erythropoietic activity such as in anaemia of chronicdisease. Anaemia of chronic disease may be associated with conditionssuch as renal insufficiency, cancer and inflammatory disorders. As notedabove, iron deficiency may also commonly occur in these disorders so itfollows that treatment through iron supplementation may address irondeficiency alone and/or anaemia of chronic disease.

EXAMPLES

Materials and Methods

Centrifugation

Recovery of agglomerates was carried out by centrifugation orultrafiltration on a Mistral 6000 centrifuge at 4500 rpm. Phasespeciation was carried out at 13000 rpm on a benchtop centrifuge.

Phase Speciation

A homogeneous aliquot (1 mL) of the suspension was collected andtransferred to an Eppendorf tube. Any centrifugable phase formed wasseparated from the solution by centrifugation (10 minutes at 13000 rpm;benchtop centrifuge). The iron concentration in the supernatant fractionwas then determined by inductively coupled plasma optical emissionspectrometry (ICP-OES). To differentiate between soluble iron andcolloidal iron (non-centrifugable particles) in the supernatant, afurther 0.7 mL aliquot was ultrafiltered (Vivaspin 3,000 Da molecularweight cut-off polyethersulfone membrane) and again analysed by ICP-OES.

TEM

The sample was prepared for TEM by dispersing in methanol anddrop-casting directly on holey carbon TEM support film (Cu-grid) andair-drying. Samples were analysed in the CM200 FEG-TEM.

XRD

Samples were crushed, and loaded in to standard plastic sample holders.The diffraction data were collected with a Bruker D8 Diffractometerusing Cu Kα radiation, employing a Vantec detector. The scanning rangewas 5-75 degrees 2θ, with a step size of 0.15°; the total time forcollection was 14 hours per sample.

Resuspension of Dried Powders

Powders produced as per examples above were resuspended in UHP water tothe initial Fe concentration (ca. 40 mM) and particle size distribution(volume based) determined by dynamic light scattering.

Lysosomal Dissolution Assay

Dissolution rates under simulated lysosomal conditions were determinedat pH 5.0±0.1 in a 10 mM citric acid, 0.15 M NaCl solution. The Fematerial was added to the assay solution at an Fe concentration of ca. 1mM and incubated for 360 min at room temperature. Phase speciation wascarried out as per above.

Example 1: Synthesis of an Iron Hydroxide Adipate Tartrate (IHAT)Suspension

2.7 g KCl, 0.90 g tartaric acid and 0.88 g adipic acid were added to abeaker containing 240 mL ddH₂O. The mixture was stirred until all of thecomponents dissolved. Then 100 mL of a ferric iron solution was added(200 mM FeCl₃.6H₂O, 0.5 mL conc. HCl in 60 mL ddH₂O). The finalconcentration of iron in the solution was 40 mM, KCl was 0.9% w/v and pHwas below 2.0. NaOH was added drop-wise (from a 5M NaOH solutionprepared in ddH2O) to this mixture, with constant stirring until7.4<pH<8.5 was achieved. This resulted in a suspension that comprisedsmall colloids (see FIG. 1) and was free of agglomerates. The processwas carried out at room temperature (20-25° C.)

Example 2: Oven Drying of an IHAT Suspension (Comparative Example)

Suspensions produced as in Example 1 were air-dried in an oven at 45° C.Drying required typically about a week (4-14 days depending on thevolume being dried). The dried material was milled by hand or micronizedwith a ball mill.

Example 3: Oven Drying of an Ultrafiltered IHAT Suspension

Reaction products, other than IHAT, and the unbound ligand fraction wereremoved from an IHAT suspension through ultrafiltration (20 mL capacityultra-filters with a PES membrane; 3000 MWCO cut off). Ultrafiltrationwas carried out by transferring 20 mL of an IHAT suspension (produced asin Example 1) and spinning at 4500 RPM until less than 2 mL of colloidalconcentrate was left. The ultrafiltrate (containing non-colloidalspecies) was then discarded, and the concentrate was diluted up to 20 mLwith ddH₂O. This suspension was ultrafiltered again at 4500 rpm and theresulting ultrafiltrate was also discarded. Finally, thecarboxylate-free colloidal concentrate was transferred to a petri dishand dried in an oven at 45° C. (3 days).

Example 4: Ethanolic Recovery

A suspension of IHAT produced as in Example 1 was diluted with ethanolat a proportion of 1:2 (15 mL IHAT+30 mL ethanol). Addition of ethanolresulted in immediate agglomeration of colloids and the resultingagglomerates centrifuged at 4600 rpm for 10 minutes. Next, thesupernatant was discarded and the pellet—containing agglomeratedIHAT—was dried for 24 hours in an oven at 45° C.

Example 5: Methanolic Recovery

The procedure was the same as in ethanolic recovery except methanol wasused.

Example 6: Acetonic Recovery

The procedure was the same as in ethanolic recovery except acetone wasused

Example 7: Synthesis of a Concentrated IHAT Suspension (200 mM Iron)

15.01 g tartaric acid and 14.61 g adipic acid were added to a beakercontaining 800 mL ddH₂O. The mixture was stirred and moderately heateduntil all of the components dissolved. Once the carboxylate solution hadreturned to room temperature, 200 mL of a ferric iron solution (54.06 gFeCl₃.6H₂O in 200 mL ddH2O) was added to it. The final concentration ofiron in the resulting solution was 200 mM and the pH was below 1.5. NaOHwas then added drop-wise (from a 5M NaOH solution prepared in ddH₂O) tothis mixture, with constant stirring until 7.8<pH<8.5 was achieved. Thisresulted in a dark suspension. The process was carried out at roomtemperature (20-25° C.)

Example 8: Ethanolic Recovery of Concentrated IHAT

The suspension of IHAT produced in Example 7 was diluted with 2 Lethanol. Addition of ethanol resulted in immediate agglomeration ofcolloids and the resulting agglomerates were centrifuged at 4600 rpm for10 minutes. Next, the supernatant was discarded and thepellet—containing agglomerated IHAT—was dried for 24 hours in an oven at45° C.

Example 9: Characterisation of Concentrated IHAT Recovered by EthanolicPrecipitation

An aliquot of the final suspension produced as in Example 7 was diluted1:5 (ca 30 mM) and characterised for particle size and phasedistribution prior to ethanolic precipitation (FIGS. 15 and 16; termed“original” in the figure legends). After ethanolically recovering andoven drying the suspension above (as per Example 8) a portion of thepowder was resuspended to ca 30 mM (0.3682 g in 50 mL water). Thissuspension was then characterised for particle size and phasedistribution (FIGS. 15 and 16; termed “resuspended” in the figurelegends), showing that the material did resuspend to its original sizeand phase distribution. Iron content of the dry powder was determined tobe 22.4±0.4% (w/w).

Results

Iron Hydroxide Adipate Tartrate (IHAT) as per previous disclosures isproduced as a suspension of small iron oxo-hydroxide colloids (FIG. 1;Example 1). These materials are ferrihydrite-based particles where themineral phase of the iron hydroxide has been disrupted by tartrateligands mostly (FIG. 2). Thus far, dry IHAT materials have been producedby simply evaporating water from the suspension (as per Example 2).However, this is a lengthy and energetically costly process. Also, asstated above, the ‘trapping’ of unbound carboxylate ligands in the finalpowder produces formulations with low iron content (i.e. large pills arerequired). Alternatively, ultrafiltration can be used to remove unboundsoluble species prior to drying (FIG. 2) and consequently increase ironcontent (Table 1) but this is also a costly strategy that cannot beeasily scaled up.

In contrast, the ethanolic-recovery process disclosed herein is a fastand cheap process that also reduces the level of the unbound speciespresent in the materials (FIG. 4) and increases their iron content(Table 1). Critically, despite the reduction in the carboxylate load,the mineral phase (i.e. ferrihydrite) of ethanolic-recovered materialsremains disrupted (FIGS. 5 to 7) and crystallites are very small (FIG.14). Mineral disruption leads to an increase in the chemical lability ofiron oxo-hydroxide materials which is linked to their ability to releasebioavailable iron. As such, adequate disruption of the mineral phase ofethanolic recovered IHAT was further confirmed through an in vitrolysosomal dissolution assay. This showed that dissolution rates ofoven-dry, ultra-filtered and ethanolic-recovered IHAT materials havesimilar properties although the materials of the present inventiondissolve more rapidly than the corresponding oven-dried ones (FIG. 8),attributed to their even smaller primary crystallite size.

TABLE 1 Iron content (as determined by ICP-OES) of IHAT materialsrecovered through different strategies. Material % Fe (w/w) Regular IHAT(Example 2) 7.45 ± 0.1 Ultrafiltered IHAT (Example 3) 37.64 ± 0.01Ethanolic recovery IHAT (Example 4) 26.5 ± 0.1

The long drying times of the existing oven drying process (Example 2)also leads to the formation of an unwanted fraction of irreversibleaggregates that do not resuspend when back in water (FIG. 9) and whichare therefore not a source of bioavailable iron. In contrast, whenre-hydrated, ethanolic-recovery materials re-suspend completely (FIG.10) to their original size (FIG. 11). This is highly surprising sincethe carboxylate load, which contributes to the stability of thesesuspensions through electrostatic repulsion, is greatly reduced with theethanolic-recovery process and in the art would normally be anticipatedas essential to disperse the particles. Therefore the method producesmaterials with several advantages over prior art IHAT materials: overallit produces even smaller primary particle sizes of the carboxylateligand modified ferric iron oxo-hydroxides which are more easilydissolved under lysosomal conditions and thus expected to be morebioavailable; it prevents aggregation and/or agglomeration of a fractionof the dried material; and the reduction in the carboxylate content ofthe materials increases the overall iron content present in thematerials on a percentage weight for weight basis.

The experiments described above also show that other water misciblenon-aqueous solvents, in particular acetone (FIG. 12) and methanol (FIG.13), can be utilised instead of ethanol.

Carboxylate Content in Ethanolically Recovered IHAT

Prior to ethanolic synthesis, IHAT (as per Example 1) comprised aligand:iron ratio of 0.5:1 for both adipate and tartrate. Analysis ofthe carboxylate content of ethanolic recovered material (produced as perExample 4) showed that the tartrate:iron ratio had only dropped to0.40:1 whereas the adipate:iron ratio had dropped to 0.09:1. Whilst notwishing to be bound by any particular theory, the carboxylate content inethanolically recovered IHAT may be indicative of a greater level ofassociation and/or modification to the oxo-hydroxide mineral by tartratethan by the adipate.

REFERENCES

The following references are expressly incorporated by reference for allpurposes in their entirety.

-   Pereira et al., Nanomedicine, 10(8): 1877-1886, 2014. doi:    10.1016/j.nano.2014.06.012-   Aslam et al., Ferroportin mediates the intestinal absorption of iron    from a nanoparticulate ferritin core mimetic in mice. FASEB J.    28(8):3671-3678, 2014.-   Powell et al., A nano-disperse ferritin-core mimetic that    efficiently corrects anaemia without luminal iron redox activity.    Nanomedicine, 10(7):1529-1538, 2014.-   WO 2008/096130.-   http://www.rsc.org/chemistryworld/2014/12/solving-iron-solubility-problem-profile-mrc-   Heinrich. Bioavailability of trivalent iron in oral preparations.    Arzeinmittelforshung/Drug Research 1975; 25(3): 420-426.-   Geisser & Müller, Pharmacokinetics of iron salts and ferric    hydroxide-carbohydrate complexes. Arzneimittelforshung/Drug    Research, 37 (1): 100-104, 1987.-   Nielsen et al., Bioavailability of iron from oral ferric polymaltose    in humans. Arzneimittelforshung/Drug Research, 44(1): 743-748, 1994.-   U.S. Pat. No. 3,076,798.-   US 2006/0205691.-   WO 2003/092674.-   WO 2006/037449.-   Harvey et al., Ferric trimaltol corrects iron deficiency anaemia in    patients intolerant to iron. Alimentary Pharmacology & Therapeutics,    12(9):845-848, 1998.-   WO 2003/097627.-   WO 2004/074444.-   US 2008/0274210.-   Bobtelsky M and Jordan J. The structure and behaviour of ferric    tartrate and citrate complexes in dilute solutions. J.A.C.S., 69:    2286-2290, 1947.-   U.S. Pat. No. 3,821,192.-   WO 2005/094203.-   WO 2005/000210.-   US 2005/0256,328.

The invention claimed is:
 1. A method of producing a carboxylate ligandmodified ferric iron hydroxide formulation, the method comprising:mixing a colloidal suspension of the carboxylate ligand modified ferriciron hydroxide in a water miscible non-aqueous solvent to cause thecarboxylate ligand modified ferric iron hydroxide to agglomerate;recovering the agglomerated carboxylate ligand modified ferric ironhydroxide; and drying the carboxylate ligand modified ferric ironhydroxide to produce the carboxylate ligand modified ferric ironhydroxide formulation; wherein the carboxylate ligand comprises one ormore dicarboxylate ligands of the formulaHOOC—R₁—COOH or an ionized form thereof, wherein R₁ is an optionallysubstituted C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl or C₁₋₁₀ alkynyl group; whereinthe water miscible non-aqueous solvent is selected from ethanol,methanol, and/or acetone; and wherein the carboxylate ligand modifiedferric iron hydroxide has a three dimensional polymeric structure inwhich the carboxylate ligands are non-stoichiometrically substituted forthe oxo or hydroxy groups of the ferric iron hydroxide so that some ofthe ligand integrates into the solid phase by formal metal-ligandbonding.
 2. The method of claim 1, wherein the three dimensionalpolymeric structure of the carboxylate ligand modified ferric ironhydroxide is such that the substitution of the oxo or hydroxy groups bythe carboxylate ligands is random.
 3. The method of claim 1, wherein themethod further comprises initial steps of mixing a solution of ferriciron ions and one or more carboxylic acid ligands or carboxylate ligandsand increasing the pH of the solution to cause formation of thecolloidal suspension of the carboxylate ligand modified ferric ironhydroxide.
 4. The method of claim 3, wherein the pH is increased by theaddition of alkali, optionally wherein the alkali is sodium hydroxide.5. The method of claim 3, wherein the pH is increased to a pH between7.0 and 9.0.
 6. The method of claim 3, wherein the pH is increased to apH between 7.4 and 8.5.
 7. The method of claim 1, wherein thecarboxylate ligand modified ferric iron hydroxide formulation comprisesadipate and tartrate ligands or tartrate and succinate ligands orsuccinate and adipate ligands.
 8. The method of claim 7, wherein theligands and ferric iron ions are present in a molar ratio of 1:1:2(tartrate:adipate:Fe), 1:1:2 (tartrate:succinate:Fe), 1:6:2(tartrate:succinate:Fe) or 1:1:2 (succinate:adipate:Fe).
 9. The methodof claim 7, wherein the carboxylate ligand modified ferric ironhydroxide formulation comprises a concentration of ferric iron between20 mM and 1000 mM, a concentration of adipate between 10 mM and 150 mM,and a concentration of tartrate between 10 mM and 500 mM.
 10. The methodof claim 9, wherein the concentration of ferric iron is between 20 mMand 80 mM, the concentration of adipate is between 10 mM and 40 mM andthe concentration of tartrate is between 10 mM and 40 mM.
 11. The methodof claim 9, wherein the concentration of ferric iron is about 40 mM, theconcentration of adipate is about 20 mM and the concentration oftartrate is about 20 mM.
 12. The method of claim 9, wherein theconcentration of ferric iron is about 200 mM, the concentration ofadipate is about 100 mM and the concentration of tartrate is about 100mM.
 13. The method of claim 1, wherein the carboxylate ligand modifiedferric iron hydroxide formulation comprises tartrate and/or tartaricacid ligands.
 14. The method of claim 1, wherein the ratio of the volumeof the water miscible non-aqueous solvent to the colloidal suspension ofthe carboxylate ligand modified ferric iron hydroxide is between 1:1 and5:1.
 15. The method of claim 1, wherein the water miscible non-aqueoussolvent is ethanol.
 16. The method of claim 1, wherein the drying steptakes 24 hours or less at 45° C.
 17. The method of claim 1, wherein thesteps of agglomerating the suspension and recovering the agglomeratedcarboxylate ligand modified ferric iron hydroxide removes unreactedferric iron ions (Fe3+), sodium chloride and/or one or more carboxylicacid ligands or carboxylate ligands from the carboxylate ligand modifiedferric iron hydroxide.
 18. The method of claim 1, wherein thecarboxylate ligand modified ferric iron hydroxide has an iron content ofat least 10% Fe (w/w), or at least 20% Fe (w/w).
 19. The method of claim1, where the carboxylate ligand modified ferric iron hydroxideformulation has a mean particle size between 1 and 10 nm.
 20. The methodof claim 1, wherein the ligand disrupts the structure of the carboxylateligand modified ferric iron hydroxide formulation as determined usingX-ray diffraction (XRD).
 21. The method of claim 20, wherein thecarboxylate ligand modified ferric iron hydroxide formulation has astructure that is consistent with ligand modified ferrihydrite collectedby oven drying, wherein the structure is determined by electronenergy-loss spectroscopy (EELS), and wherein the EELS spectrum comprisesferric iron and organic ligand-altered carbon and oxygen edges.
 22. Themethod of claim 1, further comprising a step of milling or micronizingthe carboxylate ligand modified ferric iron hydroxide formulation. 23.The method of claim 1, further comprising a step of formulating thecarboxylate ligand modified ferric iron hydroxide formulation by mixingit with one or more pharmaceutically acceptable excipients.
 24. Themethod of claim 23, further comprising making tablets or capsules. 25.The method of claim 23, wherein the carboxylate ligand modified ferriciron hydroxide formulation is formulated for oral delivery.
 26. An ironsupplement tablet, capsule or powder comprising the carboxylate ligandmodified ferric iron hydroxide formulation produced by the method ofclaim
 1. 27. The method of claim 1, wherein the optional substitutionincludes one or more hydroxyl groups.